QUADRA 96® DMV
PIPETTOR HEAD

The new Quadra 96® DMV pipettor head adds increased flexibility to Tomtec’s Quadra 4 product line.  The DMV (Disposable Multiple Volume) pipettor head will accommodate 4 different sets of pipettor tips.  A 250µL, a 50µL tip, a 20µL tip, and a 250µL tip with a wide bore.  All are available from Tomtec.  Each rack of tips are interchangeable, even within a pipetting protocol.  It allows the user to select the appropriate tip for each specific application.

The use of disposable tips requires the use of an air displacement pipettor.  The liquid aspirated in the tip must never contact the pipettor piston.  As such, the minimum dead air gap must be equivalent to the tip volume, i.e. a 250µL tip always has a minimum of 250µL of air between the liquid being aspirated and the face of the pipettor piston.

Air displacement pipettors are defined by the General Gas Law PV=ηRT.  When aspirating the same gas (air) at the same temperature, the equation becomes P1V1=P2V2.  When aspirating the maximum tip volume, the aspirating vacuum within the tip is limited to half of atmospheric pressure.  For volumes less than the maximum tip volume, the aspirating vacuum is proportionately less.  Thus, at volumes less than 10% of the tip volume, air displacement pipettors loose accuracy.  There is insufficient negative pressure to overcome, the resistance to flow within the tip itself.  These are the forces of capillary action.  Thus, the ability to choose the right tip for the volume to be dispensed, increases operational accuracy.

The Quadra 4 stacker design allows the appropriate tip rack to infeed to the shuttle and then outfeed back to the stackers.  The further ability of the Quadra 4 stacker design allows restacking, which provides the means to sort incoming tip racks, microplates, and reservoirs, as they come and go to the shuttle for processing.  The flexibility of operation is left solely to the creativity of the operator in structuring the desired protocol.

As with the incoming microplates and reservoirs, the Quadra 4 requires the database value of the tip or the other devices that are on the shuttle at any given time.  This is accomplished with the shuttle layout icon.  Each time a different plate, reservoir, or rack of tips comes on the shuttle, the shuttle layout must agree with the devices that are in place.  A change in layout is only required if a different device is used.  It is not required if the same devices is simply exchanged, i.e. one rack of 250µL tips for another rack of 250µL tips.  The database values, for each device, provides the essential properties of the device, i.e. top of well, bottom of well, tip volume, etc.

The tip racks for the DMV tips are all of a common size with the SBS footprint.  The tip volume is a database value that describes the pipetting limits.  The 250µL and 50µL tips are of the same length, however, the 20µL tip is a shorter length.  The database value provides that correction.  The 50µL tip has a longer narrow cannula.  Depending on the liquid being handled, it may be necessary to use a slower aspirate speed.  This is to assure that the liquid level in the tip is following the piston motion.  The longer cannula of the 50µL tip, may restrict the flow of more viscous liquids, such as plasma.

To increase the accuracy of air displacement pipetting at smaller volumes, the Quadra 4 provides 2 distinct pipetting features.  The first is the ability to blow through the tip with low air pressure.  This is the Quadra’s timed dispense function.  At the conclusion of the dispense motion, low positive pressure air (1 to 5 psi) may be blown through the tip orifice.  This effectively clears the tip orifice of the variable volume that may be retained by capillary action.  The 1µL, that may, or may not be retained by capillary action, may only be 1% of a 100µL dispense, but it is 10% of a 10µL dispense.  The use of the timed dispense function is far more effective at consistently clearing the tip of residual than the customary use of piston blow out air gaps.

The second function feature is target dispensing to improve accuracy at a low volume.  The target method may be selected on any aspirate/dispense function. There are two phases to the target method.  Experience and collected data has shown that pre-wetting the tip, prior to aspirating the sample volume, improves pipetting accuracy at the small volumes.  When target method is selected, the tips are automatically pre-wetted by aspirating and dispensing 30µL back to the source.  For dispense volumes less than 50µL, the second step is to aspirate 50µL then automatically dispense back to the source all but the target volume.

Assume 10µL is the target volume to be dispensed.  30µL is first aspirated and dispensed back to the source to pre-wet the tip interior.  Then 50µL is aspirated and 40µL is dispensed back to the source leaving that target volume of 10µL in the tip.

What is the purpose of this motion?  As stated earlier, by aspirating 50µL, a higher internal vacuum is created in the tip, than if only 10µL was aspirated.  This higher vacuum is more effective in filling the tip with the desired liquid aliquot by overcoming the resistance to flow at the small orifice.

Next, the pipette piston reverses to the dispense function, thereby forcing the excess volume out of the tip, leaving the requested target volume to be dispensed to the destination.

The last part of the target method pipetting occurs on the dispense at the destination.  The pipettor piston moves to dispense all but the last 3µL in the tip.  The last 3µL are then ejected with a positive pressure of 2-3 psi through the tip orifice.  The imparted kinetic energy, to the remaining 3µL slug of liquid, causes it to eject cleanly from the tip orifice.

The 3µL value was arrived at empirically.  Initial testing left 8µL to be ejected by air pressure.  There was not enough velocity applied to the liquid slug which allowed some of it to cling to the tip exterior on the dispense.  The 3µL volume gave better results for the reason stated.

The above stated pre-wet and 50µL aspirate for the target method are based on the 250µL tip.  The same value would apply when using the 50µL tip i.e., 30µL pre-wet, 50µL then dispensed to the target volume.  When the 20µL is used, the pre-wet and put back volumes would be the full tip volume of 20µL.

Creating a stacker program on a Quadra 4 is akin to a software game.  There are certain rules that apply.  The objective is to create a smooth running program that can be repeated on demand.  The real advantage of automation is to create a specific protocol, that will run precisely the same, every time.  This allows small optimizing modifications that will be faithfully reproduced to enhance the end results.  Once the program is created, anyone can execute it.  The stackers are loaded in the defined order of the program and then executed precisely the same every time the protocol is run.

The basic rules are as follows: First, an understanding of stacker access by the shuttle stations.  Shuttle station 2 is the most flexible.  It can access all four stackers.  Stations 1 can access the Left Rear Stacker (LRS) and the Left Front Stacker (LFS).  Station 3 can access the Right Rear Stackers (RRS) and the right front stacker (RFS).  Station 5 can access the LFS and RFS.  Stations 4 and 6 can only access the LFS and the RFS. Thus, they are the least useful.  Therefore, they are commonly used for fixed functions, such as the Ultrasonic Tip Wash Station or a vacuum box.  They are also useful for a common reservoir that is used through out the protocol.

While each pipetting protocol may be different, in general terms, it is effective to use the left rear stacker for the incoming source plates, or reservoirs and the right rear stacker for the destination plates.  In this manner at the end of the sequence, the source plates, or reservoirs may be restacked for the next set of destination plates.  The tip boxes are then interspersed, depending on whether they are to be assigned and reused with the specific sources or not.  A distinct advantage of the Quadra 4 is the ability to make a non-contact dispense.  The target method may be used to make a clean dispense at the top of the well, without touching the contents of the receiving plate.  This allows assigning a rack of tips for use with specific reagents, without fear of cross contamination.

As the pipetting protocol become more complex, moving microplates, reservoirs and tips in and out of the stackers to the shuttle becomes more challenging.  While everybody finds their own best way, a starting point is to step the protocol through the entire program, step-by-step using the simple Quadra programming features.  It provides a visual presentation of the program as you proceed.

The program allows you to assign a descriptive name to each plate, reservoir, or rack of tips.  This aids in tracking the program.  As each new device is placed on the shuttle, use the shuttle layout icon to bring the appropriate database values.  At the end, it is essential that you record the starting location of each microplate, reservoir, or tip rack in their respective stackers.  This information may be recorded in an initial pause statement.  

Tomtec personnel are available to assist you in creating a stacker program.